Gravitational Tempering in Colloidal Epitaxy To Reduce Defects Further

Less-defective colloidal crystals can be used as photonic crystals. To this end, colloidal epitaxy was proposed in 1997 as a method to reduce the stacking defects in colloidal crystals. In this method, face-centered cubic (fcc) (001) stacking is forced by a template. In fcc (001) stacking, in contrast to fcc {111} stacking, the stacking sequence is unique, and thus the stacking fault can be avoided. Additionally, in 1997, an effect of gravity that reduces the stacking disorder in hard-sphere (HS) colloidal crystals was found. Recently, we have proposed a gravitational tempering method based on a result of Monte Carlo (MC) simulations using the HS model; after a colloidal crystal is grown in a relatively strong gravitational field, the defects can be reduced by decreasing the gravity strength and maintaining it for a period of time. Here, we demonstrate this method using MC simulations with a programmed gravitation. The dramatic disappearance of defect structures is observed. Gravitational tempering can complement gravitational annealing; some defect structures that accidentally remain after gravitational annealing (keeping the colloidal crystal under gravity of a considerable constant strength) can be erased

First Direct Observation of Impurity Effects on the Growth Rate of Tetragonal Lysozyme Crystals under Microgravity as Measured by Interferometry

The normal growth rates <i>R</i> and apparent step velocities (lateral growth rates of a spiral hillock) <i>V</i> of tetragonal hen egg-white lysozyme (HEWL) crystals were for the first time measured by Michelson interferometry in the international space station (as part of the NanoStep project) using commercialized HEWL samples containing 1.5% impurities. A significant increase in <i>V</i> under microgravity was confirmed compared to step velocities <i>V</i>_step on the ground, while a decrease in <i>R</i> was also confirmed compared to that in the purified solution under microgravity as expected. Because of exact measurement of growth rates, kinetic analyses of <i>R</i> were conducted as a function of supersaturation, σ (σ ≡ ln­(<i>C</i>/<i>C</i>_e), where <i>C</i> is the concentration; <i>C</i>_e is the solubility), using a spiral growth model and a two-dimensional (2D) nucleation growth model. For both models over a wide range of σ, <i>R</i> in the impure solution was significantly lower than that in the purified solution. The degree of the suppression of impurity effects was also evaluated using the difference in <i>V</i>_p and <i>V</i>_i, where <i>V</i>_p is the apparent step velocity in the purified solution, and <i>V</i>_i is that in the impure solution. The difference between <i>V</i>_p and <i>V</i>_i was smaller than the difference in step velocities on the ground, <i>V</i>_step,p and <i>V</i>_step,i, where <i>V</i>_step,p is the step velocity in the purified solution, and <i>V</i>_step,i is the step velocity in the impure solution

Precipitant-Free Lysozyme Crystals Grown by Centrifugal Concentration Reveal Structural Changes

The three-dimensional (3D) structure of a protein molecule in its crystal need not correspond to that found in vivo in many cases, since we usually crystallize protein molecules using precipitants (salts, organic solvents, polymeric electrolytes, etc.), and the precipitants are often incorporated into crystals along with the protein molecules. Although precipitant-free crystallization methods would solve these problems, such methods had not yet been established. We have achieved a novel precipitant-free crystallization method by liquid–liquid phase separation during the centrifugal concentration of lysozyme in ultrapure water. In the 3D structure of the precipitant-free crystal, lysozyme loses a sodium cation and changes the position of Ser72. Deionization of the solution also appears to induce a change in the position of Asp101 and an increase in the activity of lysozyme

Precipitant-Free Lysozyme Crystals Grown by Centrifugal Concentration Reveal Structural Changes

The three-dimensional (3D) structure of a protein molecule in its crystal need not correspond to that found in vivo in many cases, since we usually crystallize protein molecules using precipitants (salts, organic solvents, polymeric electrolytes, etc.), and the precipitants are often incorporated into crystals along with the protein molecules. Although precipitant-free crystallization methods would solve these problems, such methods had not yet been established. We have achieved a novel precipitant-free crystallization method by liquid–liquid phase separation during the centrifugal concentration of lysozyme in ultrapure water. In the 3D structure of the precipitant-free crystal, lysozyme loses a sodium cation and changes the position of Ser72. Deionization of the solution also appears to induce a change in the position of Asp101 and an increase in the activity of lysozyme

Precipitant-Free Lysozyme Crystals Grown by Centrifugal Concentration Reveal Structural Changes

The three-dimensional (3D) structure of a protein molecule in its crystal need not correspond to that found in vivo in many cases, since we usually crystallize protein molecules using precipitants (salts, organic solvents, polymeric electrolytes, etc.), and the precipitants are often incorporated into crystals along with the protein molecules. Although precipitant-free crystallization methods would solve these problems, such methods had not yet been established. We have achieved a novel precipitant-free crystallization method by liquid–liquid phase separation during the centrifugal concentration of lysozyme in ultrapure water. In the 3D structure of the precipitant-free crystal, lysozyme loses a sodium cation and changes the position of Ser72. Deionization of the solution also appears to induce a change in the position of Asp101 and an increase in the activity of lysozyme

Experimental designs.

<p>The figure shows the three experimental groups of cell injection. BMSCs in PBS were injected into the CSF three times (once weekly) into the 4th ventricle 1, 2, or 4 weeks after contusion injury to the spinal cord. For the control, the vehicle (PBS) without BMSCs was injected in the same manner. Cell- or vehicle-injection started at 1 week post-injury (PI) in Group 1 (A), at 2 weeks PI in Group 2 (B), and at 4 weeks PI in Group 3 (C). Most rats were fixed at 4 weeks following the initial injection. A few rats were fixed 2 weeks after the initial injection.</p

BMSCs in the spinal cord lesion of the 1-w PI group.

<div><p>A: A few BMSCs (green) are found within the lesion 2 days after transplantation. B: The region of the rectangle was enlarged to show BMSCs (arrows) within the lesion. Astrocytes are immunostained red.</p> <p>Scale: 1 mm for (A), 250 µm for (B).</p></div

Differences in cavity formation between the BMSC-transplanted and the PBS-injected (control) rats.

<div><p>The pictures show the representative horizontal sections of the spinal cord from rats of 1-w, 2-w and 4-w PI. HE-staining.</p> <p>(A) Spinal cord sections of BMSC-transplanted (a-1) and PBS-injected (a-2) rats at 1-w PI. The cavity volume (a-3) relative to the whole spinal cord volume was 55.5 ± 5.3% (mean ± SEM) in the control, and 10.4 ± 3.5% in the BMSC-transplanted rats (* p < 0.05). R: rostral direction for all horizontal sections.</p> <p>(B) Spinal cord sections of BMSC-transplanted (b-1) and PBS-injected (b-2) rats at 2-w PI. The cavity volume (b-3) relative to the whole spinal cord volume was 50.2 ± 5.5% in the control, and 13.5 ± 2.0% in the BMSC-transplanted rats (* p < 0.05).</p> <p>b-4: Immunohistochemistry for macrophages from the spinal cord lesion of the section corresponding to the site shown in b-2. Macrophages are immunostained for OX42 on the cell surface. Scale: 100 µm.</p> <p>(C) Spinal cord sections of BMSC-transplanted (c-1) and PBS-injected (c-2) rats at 4-w PI. The cavity volume (c-3) relative to the whole spinal cord volume was 70.5 ± 5.0 in the control, and 31.8 ± 3.7% in the BMSC-transplanted rats (* p < 0.05). Scale: 1 mm for all HE-stained sections.</p></div

Locomotor evaluation.

<div><p>BMSCs were injected into the CSF three times (once weekly) at 0, 1 and 2 weeks beginning at 1-w, 2-w, and 4-w PI as indicated on the abscissa in the graph. The rats were observed for 4 weeks following the initial injection.</p> <p>(A) The BMSC transplantation was performed at 1-w PI.</p> <p>The locomotor improvement becomes obvious from 2 weeks post-initial injection (pIn). The average BBB scores reach 9.0 ±1.0 points (mean ± SEM) at 4 weeks pIn. The average BBB score of the control rats remains at 3.0 ± 0.5 points (*: p < 0.01)..</p> <p>(B) The BMSC transplantation was performed at 2-w PI.</p> <p>The locomotor improvement becomes obvious from 3 weeks pIn. The average BBB scores are 10.9 ± 2.2 points at 4 weeks pIn. The BBB score of the control rats remains at 5.0 ± 2.1 points (*: p < 0.05 at 3weeks, and p < 0.01 at 4 weeks pIn)..</p> <p>(C) The BMSC transplantation was performed at 4-w PI.</p> <p>The locomotor improvement is gradual. The average BBB scores reach 10.2 ± 1.0 points at 4 weeks pIn. The BBB score of the control rats remains at 5.1 ± 1.7 points. (*: p < 0.01 at 3 and 4 weeks pIn.)</p></div

Fluorescence density of FITC-labeled astrocytes.

<div><p>An example of a transverse section of spinal cord, in which the fluorescence density was measured, is presented in (A). This section is the same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073494#pone-0073494-g006" target="_blank">Figure 6A</a>. Fluorescence density was measured using ImageJ at 4 points (the rectangles) in the right half and 4 points in the left half of the transverse section: 2 at the near-midline on the border of the lesion and 2 on the outer surface of the spinal cord; 2 at lateral site on the border of the lesion and 2 on the outer surface of the spinal cord.</p> <p>M: midline, D: dorsal side of the spinal cord.</p> <p>The graphs show the fluorescence density of astrocytes in the 1-w (B), 2-w (C) and 4-w PI (D) groups of BMSC-transplanted subgroups, and in the 4-w PI group of PBS-injected (control) subgroup (E). There is no significant difference in fluorescence density between the border of the lesion and the outer surface of the spinal cord in any of the groups.</p></div